The standard methods for the solid phase synthesis of peptides (SPPS) on beaded resins in the normal C-to-N direction are well developed, as they must be if long peptides are to be synthesized (reviewed in M. Bodanszky et al., “Peptide Chemistry: A Practical Textbook,” Springer-Verlag, N.Y. (2d ed., 1993) and M. Bodanszky et al., “The Practice of Peptide Synthesis,” Springer-Verlag, N.Y. (2d ed., 1993)). These methods are based on attaching the carboxy terminus of an amino-protected amino acid to the resin. It essentially allows peptides to be synthesized without racemization when amino acids with Nα-protecting groups of the urethane (carbamate) type were coupled in stepwise fashion. The amino-protecting group is removed, and the next amino acid with a free carboxy group and protected amino group is then added using standard coupling conditions. Standard peptide chemistry has been used to prepare solid phase peptide libraries of tremendous diversity (H. M. Geysen et al., Molec. Immunol., 23, 709 (1986); R. A. Houghten et al., Nature, 354, 84 (1991); K. S. Lam et al., Nature, 354, 84 (1991)).
Peptide mimetic libraries (mimetic is used to indicate a modified peptide), based on the normal C-to-N direction of peptide synthesis, have been described. A peptide phosphinate library has been synthesized and used to find potent and selective inhibitors of zinc metalloproteases (J. Jiracek et al., J. Biol. Chem., 270, 21701 (1995); J. Jiracek et al., J. Biol. Chem., 271, 19606 (1996); V. Dive et al., PNAS USA, 96, 4330 (1999)). A (hydroxyethyl)amine library has also been synthesized and used to find inhibitors of the prototypical aspartyl protease Cathepsin D (E. R. Kick et al., Chem. Biol., 4, 297 (1997)).
However, combinatorial libraries of polypeptides representative of serine and cysteine proteases have not been synthesized. This can be attributed to the fact that virtually all the effective classes of serine and cysteine classes, such as peptide aldehydes (R. C. Thompson, Biochem., 12, 47 (1973)), chloromethyl ketones (CMKs) (G. Schoellmann et al., Biochem., 2, 252 (1963)), fluoromethyl ketones (FMKs) (M. H. Gelb et al., Biochem., 24, 1813 (1985); B. Imperiali et al., Biochem., 25, 3760 (1986)), and boronic acids (C. A. Kettner et al., J. Biol. Chem., 259, 15106 (1984); C. A. Kettner et al., J. Biol. Chem., 265, 18289 (1990)), are prepared by elaboration of a peptide at its C terminus. Thus, a need exists for the development of combinatorial peptide libraries representative of the serine and cysteine protease classes. Such libraries would greatly improve the ability to develop potent and specific inhibitors of these enzymes. This, in turn, requires a free C terminus of an immobilized oligopeptide, which, in turn, requires the ability to synthesize peptides in the N-to-C direction: or “reverse peptide synthesis.”
Considering the importance of such C-terminally modified peptides and peptide libraries, relatively few reports have been devoted to the development of a direct method for peptide assembly in the inverse direction. See, for example, R. J. Broadbridge et al., Chem. Commun., 1449 (1998), K. H. Bleicher et al., Tett. Lett., 39, 4591 (1998); F. Bordusa et al., Angew Chim Int. Ed. Eng., 36, 1099 (1997); R. Leger et al., Tet. Lett., 39, 4147 (1998); R. Letsinger et al., J. Amer. Chem. Soc., 85, 5163 (1969); and 85, 3015 (1963).
Merrifield et al., J. Amer. Chem. Soc., 92, 1385 (1970), used protected amino acid hydrazides as building blocks for the C-terminal elongation of peptides, followed by deprotection and subsequent reaction of the hydrazide function with nitrite allowed the next building block to be coupled by the azide method. However, the procedure is elaborate, requiring activation and coupling at low temperature with moderate yields.
Later investigations, mainly by Bayer and co-workers, have included the use of base labile amino acid 9-fluorenylmethyl esters, which were coupled with N-hydroxybenzotriazole (HOBt)/diisopropylcarbodiimide (DIC) or N-[(1H-benzotriazole-1-yl)-(di-methylamino)methylene]-N-methylmethanaminium tetrafluoroborate N-oxide (TBTU)/N-methylmorpholine (NMM). B. Henkel et al., Liebigs Ann./Recieul, 2161 (1997). In both cases, a 30-min. preactivation step with a large excess of activator (8 equiv.) was included before addition of the amino acid ester. Using TBTU/NMM extensively racemized products were obtained, whereas coupling using HOBt/DIC gave moderate racemization but led to significant formation of other byproducts.
Sharma et al. have described a few C-terminally modified tetrapeptide HIV-1 protease inhibitors, generated in the inverse direction. For example, see, R. P. Sharma et al., published PCT applications WO 93/05065 (18 Mar. 1993) and WO 90/05738 (31 May 1990) and Chem. Commun., 1449 (1998). Sharma's approach relies on coupling of amino acid tri-tert-butoxysilyl (Sil) esters. More recently, A. Johannsson et al., J. Comb. Chem., 2, 496 (2000) described a modification of the method of Sharma et al. that involves the coupling of a photolabile resin-bound C-terminal amino acid with excess amounts of amino acid tri-tert-butoxysilyl (Sil) esters, using HATU as coupling reagent and 2,4,6-trimethylpyridine (TMP, collidine) as a base. Levels of epimerization were considerably lower than those reported for other N-to-C methods, usually ca. 5% and occasionally even below 1%. However, in unfavorable cases, as in the coupling of Ile to Ser, the level was higher (ca. 20%). Also, this method requires the synthesis of sensitive zwitterionic tris (t-butoxy silylesters) and familiarity with photolytic techniques.
Thus, a continuing need exists for simple and efficient methods for the reverse synthesis of peptides and peptide mimetics, particularly for the synthesis of oligopeptide mimetic libraries useful for high-throughput drug screening.